Karyopherin α7 (KPNA7), a divergent member of the importin α family of nuclear import receptors

Center for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA.
BMC Cell Biology (Impact Factor: 2.34). 08/2010; 11(1):63. DOI: 10.1186/1471-2121-11-63
Source: PubMed


Classical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin alpha and importin beta. NLS cargo is recognized by importin alpha, which is bound by importin beta. Importin beta mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin beta triggers disassembly of the complex. To date, six importin alpha family members, encoded by separate genes, have been described in humans.
We sequenced and characterized a seventh member of the importin alpha family of transport factors, karyopherin alpha 7 (KPNA7), which is most closely related to KPNA2. The domain of KPNA7 that binds Importin beta (IBB) is divergent, and shows stronger binding to importin beta than the IBB domains from of other importin alpha family members. With regard to NLS recognition, KPNA7 binds to the retinoblastoma (RB) NLS to a similar degree as KPNA2, but it fails to bind the SV40-NLS and the human nucleoplasmin (NPM) NLS. KPNA7 shows a predominantly nuclear distribution under steady state conditions, which contrasts with KPNA2 which is primarily cytoplasmic.
KPNA7 is a novel importin alpha family member in humans that belongs to the importin alpha2 subfamily. KPNA7 shows different subcellular localization and NLS binding characteristics compared to other members of the importin alpha family. These properties suggest that KPNA7 could be specialized for interactions with select NLS-containing proteins, potentially impacting developmental regulation.

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Available from: Joshua Brian Kelley
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    • "To further characterize the interaction between PSPC1 and IMPαs from each of the three structural clades (Kelley et al., 2010), we performed an enzyme-linked immunosorbent assay (ELISA)based IMP-binding assay using affinity-purified HIS-tagged recombinant PSPC1 protein with GST-tagged or GST-void IMPα (Supplemental Figure S2, A–C). Data from a representative experimental replicate are presented in Figure 1D; mean K d and B max values from three independent experiments are shown in Table 1. "
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    ABSTRACT: Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPα proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMPαs control cellular development, we conducted a yeast-two-hybrid screen for IMPα2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPα2 expression coincident with germline masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPα2 binding partner. PSPC1-IMPα2 binding in testis was confirmed in immunoprecipitations and pull-downs, and an ELISA-based assay demonstrated direct, high-affinity PSPC1 binding to either IMPα2/IMPβ1 or IMPα6/IMPβ1. Co-expression of full length PSPC1 and IMPα2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High throughput image analysis of >3500 cells indicated IMPα2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPα2 isoform or siRNA knockdown of IMPα2 both reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPα2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Feb 2015 · Molecular Biology of the Cell
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    • "The nuclear import of cargo is mediated through diverse pathways involving the action of karyopherins, a group of at least 20 proteins in humans [20]. While karyopherin β (KPNB, importin β) family members can directly interact with some NLSs, they commonly engage their cargo indirectly through the recruitment of proteins of the karyopherin α (KPNA/importin α) family, of which there are at least 7 different members in human [21]. Karyopherin α proteins can bind a diversity of NLSs including monopartite NLSs, consisting of a single cluster of basic amino acids, bipartite NLSs consisting of multiple clusters as well as additional nonclassical NLSs [22]. "
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    ABSTRACT: The deoxynucleotide-triphosphate (dNTP) hydrolase sterile alpha motif domain and HD domain 1 (SAMHD1) is a nuclear protein that inhibits HIV-1 infection in myeloid cells as well as quiescent CD4 T-cells, by decreasing the intracellular dNTP concentration below a level that is required for efficient reverse transcription. The Vpx proteins of the SIVSMM/HIV-2 lineage of lentiviruses bind SAMHD1 and recruit an ubiquitin ligase, leading to polyubiquitination and proteasomal degradation. Here, we have investigated the importance of nuclear localization for SAMHD1[prime]s antiviral function as well as its sensitivity to the Vpx protein of SIVMAC. Using GST pull down assays, as well as RNA silencing approaches, we show that SAMHD1 preferentially uses karyopherin alpha2 (KPNA2) and a classical N-terminal nuclear localization signal (14KRPR17) to enter the nucleus. Reduction of karyopherin beta1 (KPNB1) or KPNA2 by RNAi also led to cytoplasmic re-distribution of SAMHD1. Using primary human monocyte-derived macrophages (MDM), a cell type in which SAMHD1 is naturally expressed to high levels, we demonstrate that nuclear localization is not required for its antiviral activity. Cytoplasmic SAMHD1 still binds to VpxMAC, is efficiently polyubiquitinated, but is not degraded. We also find that VpxMAC-induced SAMHD1 degradation was partially reversed by ubiquitin carrying the K48R or K11R substitution mutations, suggesting involvement of K48 and K11 linkages in SAMHD1 polyubiquitination. Using ubiquitin K-R mutants also revealed differences in the ubiquitin linkages between wild type and cytoplasmic forms of SAMHD1, suggesting a potential association with the resistance of cytoplasmic SAMHD1 to VpxMAC induced degradation. Our work extends published observations on SAMHD1 nuclear localization to a natural cell type for HIV-1 infection, identifies KPNA2/KPNB1 as cellular proteins important for SAMHD1 nuclear import, and indicates that components of the nuclear proteasomal degradation machinery are required for SAMHD1 degradation.
    Full-text · Article · Apr 2014 · Retrovirology
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    • "The importin α gene family has undergone considerable expansion during the course of eukaryotic evolution. Human importin α is a helical molecule with at least seven isoforms [13], [14], [15], which are categorized into three phylogenetically distinct subfamilies (α1, α2 and α3) based on their amino acid sequence similarity. Subfamily α1 includes importin α5 (NPI-1), importin α6 (KPNA5), and importin α7 (KPNA6). "
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    ABSTRACT: The nucleoprotein (NP) of influenza A virus is transported into the nucleus via the classical importin α/β pathway, and proceeds via nuclear localization signals (NLSs) recognized by importin α molecules. Although NP binds to importin α isoforms Rch1, Qip1 and NPI-1, the role of each individual isoform during the nuclear transport of NP and replication of the influenza virus remains unknown. In this study, we examined the contribution of importin α isoforms for nuclear localization of NP and viral growth using a panel of NP mutants containing serial alanine replacements within an unconventional NLS of NP. Alanine mutation at amino acid 8 (R8A) caused a significant reduction in the nuclear localization and binding to the three importin isoforms. The R8A NP mutant virus did not generate by reverse-genetics approach. This indicates that position 8 is the main site that mediates nuclear localization via interactions with Rch1, Qip1 and NPI-1, and subsequent viral production. This was confirmed by the finding that the conservation of amino acid 8 in human- and avian-origin influenza virus NP was necessary for virus propagation. By contrast, another mutant, S9A NP, which localized in the nucleus, caused a reduction in viral growth and vRNA transcription, suggesting that the unconventional NLS within NP may be associated with nuclear transport, vRNA transcription and viral replication through independent pathways. Interestingly, the N-terminal 110-amino acid region, which contained the unconventional NLS with S9A mutation, mainly bound to Qip1. Furthermore, activities of vRNA transcription and replication of S9A NP mutants were decreased by silencing Qip1 in without changing nuclear localization, indicating that Qip1 involves in multiplication of S9A mutant virus independently of nuclear transport function. Collectively, our results demonstrate the unconventional NLS within NP might have the additional ability to regulate the viral replication that is independent of nuclear localization activity via interactions with Qip1.
    Full-text · Article · Sep 2013 · PLoS ONE
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